Immunoassay products and processThe application is based on Application No. 201280022626.1, the original application that the applying date is on May 10th, 2012The divisional application proposed.
Technical field
Embodiment disclosed herein is related to equipment and process for detecting and positioning the material included in blotting membrane.MoreEspecially, it is related to a kind of technology for being used to apply reagent, washing lotion and detection chemistry to blotting membrane, with via vacuum or malleationUsing and rapidly realize this detect.
Background technology
The use of gel electrophoresis is currently the common technique of the separation for biomaterial.Non-biological material can also be usedGel or other chromatograms support to separate, but bigger relative to the effort scope of biological agent.Typical application includes sequenceDetermine under background；In polymorphic detection；Or the separation of the nucleic acid fragment of the various sizes in the size verification under other backgrounds.What is also continually performed is that protein, glycoprotein, the separation of protein fragments and gel are separated as uniformity or degree of purityThe application that checking, the identification of translation post-modification and molecular weight confirm.
In all these programs, the mixing sample of biological entities is applied in running gel, and by being applied across gelAdded electric field separates each component.Regardless of the mode for being used for making gel develop, it is necessary to carry out testing result production in some wayThe pattern of the raw migration comprising material in the sample.
In order to perform this detection, generally, gel support body is set to be contacted with marking film, material is appeared on gel with itIdentical patterns are transferred to the marking film.Then at least through detecting that " spot " is non-to reduce with protein or cleaning solution barrier filmSpecific bonding（Otherwise it will cause high-caliber noise and low-level detection）.Typical blocking agent includes casein, ox bloodPure albumen（BSA）, trometamol salting liquid with TWEEN surfactants（TBS-T solution）In skimmed milk power（OneAs about 1-5%）Or the phosphate buffered saline with TWEEN surfactants（PBS-T solution）.Then with filmAntigen institute specific antibody cultivates biological entities.Then film is comprehensively washed to remove any pollutant, uncombined blocking eggWhite matter or antibody etc..Then with Primary antibodies institute specific two grades of enzymes, radio isotope, fluorine（fluorfluor）Or biotinCoupled antibody handles and cultivated film.Then film is comprehensively washed to remove uncombined secondary antibody.Then, detection examination is appliedAgent, is typically color development, chemiluminescence, fluorescence, radioactivity or streptavidin label material, and it is attached to either enzyme coupling agentCulture medium.Finally, determined using appropriate detection device the presence of biological entities, be not present, position, amount etc..Last sixStep is typically taken from from 3-6 hour to whole night, and this depends on selected reaction speed between reagent, film and biological entities.ShouldRange request film is crossed in the multiple cultivation periods mixed on platform shaken or other are appropriate.It is that most of researchers do not likeLengthy process and consumption（Waste）Substantial amounts of reagent.
Some researchers are it has been proposed that using the capillarity of absorber material, be such as placed on the filter below filmPaper, by film sucked away fluid and to improve the speed of the process, especially washing step.
US 5,155,049 refer to a kind of by being referred to as that Hoefer Scientific Instruments companies sellThe system of Hybrid-Ease hybridization chambers.This room includes two grids that film is sandwiched between it.Flase floor, which is engaged to, to be surroundedThe position of film, and the syringe being coupled in the open space produced by grid.One syringe be used to apply reagent andWash, and another is used for withdrawing margin.The substantial amounts of liquid of the system requirements, to be operated, is trouble using getting up, and be still relatively time consuming.It is also mentioned that in some special tests such as ELISA is tested, in small size trap（It is allSuch as 96 hole microtiter plates）In, other people draw liquid in washing step using vacuum by film.However, this makes itMake great efforts give a discount because its only in low volume applications can with and be still uncontrollable.They alternatively advise better methodBe used at the top of filter paper and coating on there is film and then compress be sandwiched in the film between two plates and passed through with extruding liquidFilm and the hand press entered in paper.
In the USSN 60/732,994 that on December 3rd, 2005 submits, it has been proposed that people use what is formed by multiple layersEquipment, it is included in the porous support layer below one or more layers blotting membrane, the flow distributor on blotting membrane and in streamAmount distributor on by liquid be included in desired region and allow this class I liquid I relatively low initial volume trap.Preferably, the streamIt is non-binding or low combination perforated membrane to measure distributor.
It is open in the co-pending U.S. Serial No 11/582,727 and 11/582,599 submitted on October 18th, 2006A kind of equipment, wherein, keeping the retainer and porous supporting body of fluid guider can be coupled in manifold equipment to handleVarious fluids simultaneously detect biological entities.The manifold has with and without the lid of trap and with the central opening of retainer into alwaysThe central opening of line is although useful, but its limitation that its generally uses and received with suppression.
It is apparent that there is a need of a kind of more efficient method for being used to detect biomaterial or entity on blotting membrane.It is disclosed hereinEmbodiment allow blotting membrane in biological entities more effective and efficient detection.
The content of the invention
According to some embodiments, there is provided immune detection system and method.The system includes being suitable for and such as vacuumThe manifold base of driving force connection.The manifold keeps carrier, carrier supported blotting membrane retainer.Blotting membrane retainer keeps albumenThe blotting membrane that matter can be incorporated into via electrophoresis.The system and method allow users to quickly and efficiently print proteinMark is ready for detection, such as passes through chemiluminescence.
There is provided a kind of equipment useful when performing method disclosed herein in one embodiment.The equipment includesBlotting membrane retainer（Also referred to as trace retainer, or be simply referred as retainer）, blotting membrane retainer is by lower open supportLayer and upper flow distributor are formed.Both are by such as with hinge, holder, elastic webbing, adhesive, ball-and-socket, latch and pitMethod or cooperation jointing fastener or other such means be held togather.Retainer is opened and one or more printsMark film is placed between lower floor and upper strata.Then retainer is closed and is placed to the carrier formed by upper plate and lower plateIn, the upper plate and lower plate each have at least one opening, and the opening allows fluid from the upper plate of carrier, by keepingDevice and the film being contained within, and then pass through the lower plate of carrier.Then carrier is arranged in manifold equipment to handle printSample on mark film.When the process is completed, carrier is removed from manifold, is opened, and blotting membrane retainer is removed.SoAfterwards, blotting membrane retainer is opened and blotting membrane is extracted for detected downstream.
In another embodiment, retainer is formed by the material selected from the group being made up of plastics and paper, and the retainer hasTop and bottom, each top and bottom are with the thickness between outward flange, top surface and bottom surface and top surface and bottom surface, the portionAt least one in point has from the inside solid section in outward flange, and at least top has from the inside opening in outward flange, the bottom of atThe porous supporting body formed on the top surface in portion and the flow distributor formed on the lower surface at top, distributor covering topThe opening in portion, wherein, releasedly by first and second when holder has for being aligned at the top and bottom of retainerThe device that interfixes of part so that the bottom surface at top and the top surface of bottom are aligned and arranged by mutually adjacent so that topTogether with bottom.
In another embodiment, retainer is formed so that each perforation having along side in top and bottom, andAnd the perforation is aligned on the top and bottom, and the perforation is easy to be torn to open retainer to fetch blotting membrane.
In one embodiment, carrier has outer perimeter wall, and outer perimeter wall extends with shape from the opening upwards of carrier top plateInto trap to keep reagent and wash fluid.
In another embodiment, whether at least carrier has one or more level indicators to indicate the top surface of carrier edgeIts length and preferably along its length and width level in a horizontal plane.The level can also be on manifold.
In another embodiment, level indicator is single 360 degree of bubble gauges.
In another embodiment, level indicator is at least two line horizontal bubble indicators, and at least one is by along topThe length of the top surface of plate is arranged and at least one is set by the width of the top surface along top plate.
In another embodiment,（It is one or more）Carrier inner surface is included print one or more of retainerMark film stretches to avoid forming fold and the feature for making the surface of film as flat as possible or planarizing in film.
In another embodiment, carrier has the flange at least one in the inner surface positioned at top plate or bottom plate closeSeal the stretching, extension feature formed.Preferably, it has the flange seal at least formed in the adjacent inner surface of bottom plate.More preferablyGround, the width and length that flange seal is positioned is more than the opening of retainer but less than the outer peripheral width of retainer and length.
In another embodiment, carrier has by the another of the compressible or flexible member in the half of carrier and carrierThe stretching, extension feature of relatively raised rigidity characteristic composition in half.
In one embodiment, manifold is subdivided into two or more sub- traps with parallel work-flow blotting membrane or a traceThe subdivision of film, each film is generally used to less a kind of different agent treatment.
In another embodiment, carrier is subdivided into two or more sub- traps, and retainer is also subdivided into corresponding sub- trap.
In another embodiment, the sub- trap of carrier is individually sealed and is aligned to prevent trap with the sub- trap of corresponding retainerBetween fluid migration.
In another embodiment, manifold has levelling pin, when one or more levels of the one embodiment for combining carrierIndicator comes in use, levelling pin allows manifold and carrier level in a horizontal plane.
In one embodiment, manifold has a series of pin, and it can be adjusted along vertical direction, so that when carrier quiltRelative on carrier when attaching to manifold（It is one or more）Level indicator makes manifold level.
In another embodiment, a series of pin have in screw, the threaded portion of screw fit to manifold bottom portion withPin is allowed individually to be raised or declined along vertical direction.
In another embodiment, manifold is subdivided into two or more sub- traps with parallel work-flow carrier and different blotting membranesOr the subdivision of a blotting membrane, each film is generally used to less a kind of different reagent to handle.
In another embodiment, carrier plate top openings have lid, and lid is located on the top edge of opening so that for a long time, i.e.Evaporation during incubated overnight is minimized.
In another embodiment, lid has resealable relation with trap.
In another embodiment, lid is shading, is such as made up of opaque material.
In another embodiment, the opening in carrier base has lid to prevent fluid from leaking.
In another embodiment, lid is releasedly sealed.
In another embodiment, lid has valve feature.
In another embodiment, valve has duckbilled or umbrella construction.
In another embodiment, valve available pressure difference is operated and closed when pressure differential being not present on valve.
In another embodiment there is provided it is a kind of to detect one or more biological entities on blotting membrane it is quick,Efficient and convenient method.The detection can relate to position, property or the amount of the biological substance on film.This method is related to selected from malleationOr the pressure assistant system of vacuum（regiment）, for supplying reagent to blotting membrane and removing reagent from blotting membrane, andAllow the matter detergent pollutant from film is embedded into, it will be detected using the liquid and reagent of much lower amounts.This methodMake it possible to complete to block in about 30-45 minutes in the case where not damaging trace quality, wash and antibody binding step.PeopleSimply obtain retainer, open it and on one of surface place（It is one or more）Blotting membrane so that when the equipment（It is one or more）The lower surface of blotting membrane is adjacent to porous supporting body when being closed around film and the upper surface of blotting membrane is adjacentIt is bordering on flow distributor.The equipment is placed on the manifold with pressure or vacuum source or interior, and the process starts.
The another object of embodiment disclosed herein is to provide a kind of pressure for being used to perform one or more traces or trueThe equipment that empty skeptophylaxis is determined, the equipment includes vacuum manifold, is designed to coordinate the carrier on manifold and is designed toThe device of one or more of retainer and collection antibody of the cooperation in carrier to handle trace.
The another object of embodiment disclosed herein is to provide a kind of for exempting to one or more films execution vacuum aidedThe process that epidemic disease is determined, the process includes step：
A. provide vacuum manifold, the carrier for keeping retainer, the retainer for one or more blotting membranes, include willThe one or more films for the one or more biological entities tested, the carrier is formed by top plate and bottom plate, top plate and bottomPlate each has through at least one opening of its thickness formation to allow fluid to flow there through, and the retainer, which can be located at, to be carriedIt is in fluid communication between the top plate and bottom plate of body and with least one opening of each plate of carrier, the retainer is by being maintained at oneThe porous supporting body and flow distributor risen is formed, described（It is one or more）Film is placed on porous supporting body, flow pointOrchestration is located on the top of film and retainer is placed between the top plate of carrier and bottom plate so that flow distributor is adjacent to topThe bottom of plate or inner surface and porous supporting body are adjacent to the inside of the top surface of bottom plate,
B. one or more reagents are added at least one opening of carrier top plate and applying vacuum is with by top plateThe opening of opening, the flow distributor of retainer and porous supporting body and bottom plate pulls in reagent in film, and
C. to one or more traps one or more detergent are added and applying vacuum is to pull detergent and any uncombinedIn opening and entrance vacuum manifold that reagent passes through plate top openings, flow distributor, film and porous supporting body and carrier base,And
D. according to the expectations or needs by step（B and c）It is one or many that repetition is added.
In certain embodiments, a kind of equipment for performing immunoassays is disclosed, the equipment is included by selected from by mouldingThe retainer of the material formation for the group that material and paper are constituted, the retainer has top and bottom, and each top and bottom have outerAt least one in thickness between edge, top surface and bottom surface and top surface and bottom surface, the part is with inside from outward flangeSolid section, at least top have the porous supporting body that is formed from the inside opening in outward flange, on the top surface of bottom andOpening at the top of the flow distributor formed on the lower surface at top, distributor covering, wherein, holder has for protectingThe device that the first and second parts releasedly interfix when being aligned at the top and bottom of holder so that the bottom surface at topBe aligned with the top surface of bottom and it is mutually adjacent arrange so that top and bottom are together.
In certain embodiments, outside can be included in for releasedly fixed device to be formed and around open supportThe encapsulant of body opening.The top and bottom of retainer can be made up of single piece of material, and retainer can have folding linePortion, folding line portion extends the width of retainer to form Part I and Part II.The folding line portion can be hinge.
In certain embodiments, flow distributor can have lower surface and upper surface, and the upper table of flow distributorFace can be attached to the bottom surface at the top of retainer so that on the upper surface of the thickness formation flow distributor of Part IOne or more traps.The opening at top is centrally located in the outward flange at top.
In certain embodiments, for performing the load that the equipment of immunoassays can be including retainer and for retainerBody, the retainer is formed by the material selected from the group being made up of plastics and paper, and the retainer can have top and bottom, eachTop and bottom can have in outward flange, top surface and bottom surface and the thickness between top surface and bottom surface, the part extremelyFew one can have from the inside solid section in outward flange, and at least top can have from the inside opening in outward flange, the bottom of atThe porous supporting body formed on the top surface in portion and the flow distributor formed on the lower surface at top, distributor covering topOpening, wherein, retainer can have that be used for can by the first and second parts when being aligned at the top and bottom of retainerThe device that interfixes of release ground so that the bottom surface at top and the top surface of bottom be aligned and it is mutually adjacent arrange, so as to push upPortion is together with bottom.
In certain embodiments, the equipment can also include the carrier for retainer being made up of top plate and bottom plate, topPlate and bottom plate each have width and length, top surface and bottom surface and thickness between top surface and bottom surface, outward flange and at leastOne opening, the plate has a length and width of the length and width more than retainer, the top plate of carrier have in width andThe open-topped opening of retainer is substantially equal in terms of length, the bottom surface of top plate and the top surface of bottom plate each have in two platesThe one or more sealings being mutually aligned when mutually adjacent, and the sealing is by with more than plate top openings but less than outside retainerOn each surface, at least one in plate has the outward flange that is adjacent to plate and from the plate to width and the length arrangement of sizeSeal the sealing being outwardly formed, and the device for top plate and bottom plate to be releasably held together.
In certain embodiments, the sealing in the bottom surface of top plate and the top surface of bottom plate can be flange seal.
In some embodiments it is possible in the presence of at least one the horizontal instruction equipment for the upper surface for being attached to top plate, andAnd it can include 360 degree of indicators.There may be two level indicators, one along the length of the top surface of top plate and anotherThe width of the individual top surface along top plate.
In certain embodiments, a kind of equipment for performing vacuum aided immunoassays is disclosed, the equipment includes trueEmpty manifold and the retainer formed by plastics or paper, the retainer have folding line portion, and the width of folding line portion extension retainer is with shapeInto Part I and Part II, each part has the opening being aligned when retainer is folded closure along folding line portion, manyThe first opening of hole supporter covering and the opening of flow distributor covering second, wherein, the holder has on retainer edgeThe device that the first and second parts releasedly interfix when folding line portion is folded closure, so that the first and second partsTogether.Flow distributor can be film.
In certain embodiments, a kind of equipment for performing immunoassays is disclosed, the equipment is included with baseVacuum manifold, the base has upper support surface and the gutter below supporter（drain）, upper support surface is for supportingComprising by one or more carriers of retainer to be processed, upper surface include extend through therefrom it is one or more inHeart opening, and one or more of central openings and one or more carrier alignments.
Brief description of the drawings
Fig. 1 is the exploded view of the carrier according to some embodiments；
Fig. 2 is the perspective view of the carrier in an open position according to some embodiments；
Fig. 3 is the perspective view of the carrier in the close position according to some embodiments；
Fig. 4 is the perspective view shown in cross section of the carrier according to some embodiments；
Fig. 4 A are the perspective views shown in cross section of the carrier with umbrella valve according to some embodiments；
Fig. 5 is the perspective view of the blotting membrane retainer including blotting membrane according to some embodiments；
Fig. 5 A are the perspective views of the blotting membrane retainer for showing flow distributor according to some embodiments；
Fig. 5 B are from the perspective view in terms of bottom side according to the blotting membrane retainers of some embodiments；
Fig. 6 is the perspective view of the carrier in an open position of the retainer including Fig. 5 according to some embodiments；
Fig. 7 is the decomposition diagram of the bottom side of the carrier according to some embodiments；
Fig. 8 is the perspective view of the bottom side of the carrier under the lid seated position according to some embodiments；
Fig. 9 is the perspective view of the manifold according to some embodiments；
Fig. 9 A are the perspective views of the manifold with collection vessel according to some embodiments；
Figure 10 is the decomposition diagram of the manifold including carrier according to some embodiments；And
Figure 10 A are the perspective views of the manifold, retainer and carrier according to some embodiments.
Embodiment
Fig. 1 is tuning firstly to, the carrier 10 according to some embodiments is shown.In an illustrated embodiment, carrier 10 includesLid 11, carrier top plate 12, carrier base 13, lid 14 and the air-bubble level 15 on carrier top plate 12.According to some realitiesExample is applied, carrier 10 is used to keep trace retainer flat, and conveys such as antibody, washing buffer or the like to trace retainerReagent.According to some embodiments, carrier 10 is and manifold base（It is discussed below）Separated individual devices, it allows user sameWhen multiple traces are set.Therefore, each carrier 10 can be selectively loaded the trace retainer comprising trace, add its eachFrom antibody, and then cultivated.
Fig. 2 shows the embodiment of carrier 10 in an open position, the left side in figure show the bottom side of top plate 12 andThe top side of bottom plate 13 is shown on right side.In an illustrated embodiment, top plate 12 and bottom plate 13 are hingedly connected at 16.Such as at thisShown in embodiment, hinge is the " activity for being partially bonded together two（live）" hinge.Alternatively, can individually it makeMake hinge and be attached using adhesive, thermal or machanical fastener.Other embodiment is without using hinge（It is not shown）AndIt is using holder, elastic webbing or cooperation jointing fastener（Such as slot and brake）, frictional fit pin etc., pushed up accordinglyIn portion and bottom or in be formed to and hold it together during use.Other suitable devices are for those skilled in the artIt will be apparent, and be also intended to include them.
According to some embodiments, carrier top plate 12 can have one or more traps 22, its can during use by withIn holding and/or conveying wash fluid and reagent.（It is one or more）Trap 22 can be formed with upstanding wall 22a, 22b,22c and 22d（Fig. 1）Carrier top plate 12 top surface a part, or as discrete item, it is simply attached or is placed onOn the top of carrier top plate 12.Lid 11 can be placed over the carrier 10 to cover trap 22 and prevent antibody and other fluids in extensionThe cultivation period during, particularly evaporated during incubated overnight.
Carrier base 13 includes porous supporting body 4, and it can be simple sieve, grid, flow guide grid or sinter manyThe millipore filter of pore structure, such as POREX films or thick or macropore, such as weaving or non-woven paper, polypropylene or polyethyleneFabric, glass isolator or paper or 1-10 micrometer Millipore filters.Such supporter can be by polymer, glass, ceramics or metalMaterial is made, and includes but is not limited to the metal such as stainless steel or steel alloy, aluminium and such as polyethylene, polypropylene, polysulfones, poly-The polymer such as ether sulfone, styrene, nylon.Fig. 2 shows the porous supporting body 4 of the form of flow guide grid, and it includes one and isRow groove 72 and opening 74.Opening 74 is located at the circumference inward position from porous supporting body 4.Opening 74 connects with the fluid of groove 72It is logical so that fluid is collected in groove 72 and is directed through opening 74.Groove 72 collects used fluid and will be usedFluid is transported to opening 74, and fluid is directed to waste compartment or collection tray by it（It is not shown）.If researcher wishes to collectOne or more in fluid, then suitably can do collection tray is so positioned.Those skilled in the art will recognize thatTo be that can use the groove or opening of other patterns, since it is desirable that result be by used fluid be directed to opening or oneSeries of openings, used fluid is directed to intended destination by it.
The outward flange of carrier top plate 12 and supporter 4 can be by being made with the identical material of supporter 4.When using monoblock typeDuring hinge, it must be by impact modified materials such as polyethylene, polypropylene, the flexible material of elastomer or ABS, K resinsOne in material is made.When using single hinge, holder, elastic webbing, adhesive film or other fixing devices, it can rootIt is made up according to expectation of metal, plastics or elastomer.
According to some embodiments, the first flange seal 30A is located on the upside of carrier top plate 12 as shown, andThe the second flange seal 30B that can be positioned with the porous supporting body 4 in carrier base 13 cooperate, when top plate and bottom plate are in traceFluid tight system is maintained when in the close position in the case that retainer is engaged.Therefore, flange seal 30A and 30B is determinedPosition into causing when carrier top plate 12 is rotated to closing position relative to carrier base 13, two sealings mutually registration or be aligned withCarrier is sealed.In certain embodiments, each flange seal 30A, 30B are preferably by such as silicones or thermoplastic elastomer (TPE)（TPE）Flexible elastomer be made, and be V-arrangement, a V supporting leg is shorter than another.The long end of each V-arrangement flange sealIn the respective dimple for being seated in carrier top plate and bottom plate（Fig. 4）.The short supporting leg of each v-seal is raised, can be easily deformedFeature.The shorter supporting leg of V on carrier closure and sealing station, carrier top plate will contact the top surface of trace retainer.CarryThe shorter supporting leg of V on body bottom plate will contact bottom and the deflection of trace retainer, as being seen best in Fig. 4.SealingTilt geometry in deflection applying power to pull retainer 50 or stretch it, its " tap（drum）" film and keep itIt is flat.Planar film avoids that the deformation of pinnacle and recess, the pinnacle and recess aggregation antibody volume may be produced on the surface of the film,This so cause trace diaphragm area more or less be exposed to antibody, cause bad and inconsistent result.This sealing prevents leakageAnd make it possible to realize for a long time（For example, whole night）Cultivate.Sealed design also allows needed for low power sealing mechanism and carrier 10Low chucking power.This helps the flatness for maintaining retainer and blotting membrane.Releasable breech lock 35 or other locking mechanisms are by carrier latchIt is scheduled on closing position（Fig. 3）.According to some embodiments, single flange seal can be used, the flange for being preferably used for bottom plate is closeSeal 30B.Base flange sealing 30B is used to stretch trace retainer.Once being extended, interface is designed such that carrier topTrace retainer is clamped with bottom in place.Outward flange sealing 300A, 300B are also show in Fig. 4.
Fig. 5,5A and 5B show the retainer 2 according to some embodiments.Retainer 2 includes framework 40, and framework 40 hasIt is configured to the perimeter edge of nebulin matter blotting membrane 45.In certain embodiments, framework 40 is made up of plastics or paper.At certainIn a little embodiments, the material of framework 40 can be bio-resin, cardboard, HIPS, paper or support membrane and its can be kept flatAny other appropriate rigid material.According to some embodiments, the flow distributor 46 of such as GVPP films is laminated or attached to frameFrame, and blotting membrane 45 can be positioned face thereon.Flow distributor 46 is attached to framework, and when trace retainer quiltDuring closure, blotting membrane is sandwiched between flow distributor 46 and porous supporting body 47.Porous supporting body 47 on trace retainerSupported and contacted with the porous supporting body 4 of carrier base.Flow distributor 46 is used to make blocking solution and antibody equably divideCloth is on blotting membrane.It also adjusts the flow of these solution in applying vacuum.Retainer 2 also includes porous supporting body 47, allOne piece of polypropylene non-woven grid of framework is such as incorporated into along an edge, it is allowed to which user is provided easy access to add blotting membrane,And it supports blotting membrane and prevents it to be damaged under a vacuum force.Supporter 47 is porous to allow liquid to protect by traceThe free-flowing of holder 2.Figure 6 illustrates the retainer 2 being suitably positioned in carrier 10.
In certain embodiments, retainer can be made by the way that contact adhesive is laminated into the back side of polypropylene lattice2 so that after blotting membrane is added to retainer, user removes backing strip with exposed pressure sensitive adhesive, and then by gridIt is sealed to flow distributor（For example, GVPP films）To produce sealing big envelope.In order to remove blotting membrane, it may include perforation and slide fastener withTear big envelope.
In certain embodiments, other thin slice paper materials of thin Vegetable paper, paraffin paper or paper material are laminated to polypropyleneThe side of grid.When retainer is soaked with water（As required in the process）, the paper backing of wetting adheres to GVPP films, producesTemporary sealing.After the process was complete, blotting membrane is easily removed without tearing anything or the viscous adhesive of processing.ProtectThen holder is removed, because after the drying of paper backing, it is crimped and retainer becomes unusable.
Fig. 7 and 8 illustrates the bottom side of carrier base 13.In the figure 7 it is seen that multiple openings in porous supporting body 474.Lid 14 is used to covering opening to prevent the antibody of nurturing period from leaking, such as whole particularly when long-time is cultivatedNight, as seen in fig. 8.According to some embodiments, opening 74 is surrounded by annular protrusion ring 49, and lid 14 has by structureCause the respective annular slot 60 coordinated with annular ring 49（Fig. 4）, and therefore lid can be forced on ring to be fixedTo carrier board 13.
Alternatively, lid 14 can be cancelled, and umbrella valve 144 can be used to be kept in the nurturing period in fluid, such as Fig. 4 AIt is shown.When vacuum is unlocked, valve is opened and fluid flows out.Preferably due to EPDM（Propylene diene system monomer）It is hardSpend and can rapidly be closed when vacuum is closed, valve is made up of EPDM.This helps prevent back pressure and air to carry secretly, back pressureWith air entrainment（In the case of silicone valve）Cause trace retainer to bend and cause unfavorable antibody aggregation.Antibody aggregation（Uneven covering）Region near the uneven signal for causing final trace to read, entrainment air will have weak signal（MouldPaste）And the region with aggregation antibody will have strong signal（Eye-catching）.
As shown in figs. 9 and 10, according to some embodiments, manifold 8 is can be attached to appropriate vacuum source（Do not showGo out）Vacuum manifold.Manifold 8 can include waste collection（It is not shown）, the container such as in manifold（It is not shown）To collect the liquid for being drawn through carrier 10.
According to some embodiments, manifold 8 has base 42, and it has gutter and support surface 44, one or more to carryBody 10 is placed on support surface 44.Support surface 44 includes one or more traps or carrier receiving area 55A, 55B（In figureTwo are shown in 9）, it can include the bottom side of received vector bottom plate 13 to be maintained at carrier 10 in manifold 8 with stationary modeMultiple upright ribs 56.It also show the optional corresponding controlling organization 62 for managing and monitoring manifold 8 and the process.IfIf expecting, the equipment can be used together with automatic fluid carrier etc..Manifold 8 be subdivided into one or more traps orIn the case of carrier receiving area, manifold can handle more than one retainer, with parallel work-flow carrier and different blotting membranesOr the subdivision of a blotting membrane, generally handle each film with least one different reagents.Manifold 8 can have common pressureSource, or Stress control individually can be carried out to each station carrier receiving area.
In another embodiment, manifold has levelling pin, and levelling pin works as one or many of one embodiment with reference to carrierAllow manifold and carrier level in a horizontal plane when individual level indicator is to use.In one embodiment, manifold is with oneRow pin, it can be adjusted along vertical direction, so that when carrier is attached to manifold relative on carrier（One or manyIt is individual）Level indicator makes manifold level.In another embodiment, a series of pin have screw, screw fit to manifold bottomTo allow pin individually to be raised or declined along vertical direction in the threaded portion in portion.
Various methods can be used in embodiment disclosed herein.Key factor is that they all rely on the vacuum of liquidOr malleation driving filters to approach the big inner surface area of film, it is allowed to throughout the 3D interactions of all molecules of entire depthRather than the only 2D interactions as occurred in the past at surface.In the case of using malleation, base can be canThe sealing chamber of carrier is accommodated, and lid 11 becomes pressure manifold to apply pressure to the trap 22 of carrier.
Most straightforward procedure is to perform one or more of wash cycle using presently disclosed embodiment.Generally,Each wash cycle is made up of one or more washing steps.Usually, 2-5 step is each recycled.
Another method is that embodiment disclosed herein is used in each step for needing to make liquid move through blotting membrane,Such as after the cultivation of antibody or in washing step.
During all these, it can be used and be suitable for making（It is one or more）Liquid is through equipment and enters appointing for manifoldWhat driving force.This can be according to the film and used manifold, the desired speed of filtering and researcher for being selected for traceAvailable vacuum or positive pressure source and change.
Usually, vacuum available can be in 100 and 760 mmHg（133 millibars and 1013 millibars）Between change.Valve, pressureThe use of limiter etc. can also be used to that vacuum is maintained in allowed band for used film.One embodiment it is preferredVacuum manifold uses about 100 mmHg vacuum.Other appropriate vacuum manifolds include but is not limited to can be from MassachusettsMULTISCREEN and MULTISCREEN that Billerica Millipore companies obtainHTSVacuum manifold.
Usually, airline under pressure of the malleation in the range of from about 2 psi to about 15 psi is supplied.Valve, pressureThe use of force-limiting device etc. can also be used to that pressure is maintained in allowed band for used film.Such pressure system bagInclude but be not limited to the Amicon agitating units equipment that can be obtained from Massachusetts Billerica Millipore companies andThe positive press filtration unit that can be obtained from Massachusetts Hopkinton Caliper Life Sciences companies.
In order that with the equipment according to embodiment disclosed herein, people simply obtain retainer, open it and incite somebody to action（It is one or more）Blotting membrane is placed on one in surface so that when equipment exists（It is one or more）It is closed around filmWhen, the lower surface of blotting membrane is adjacent to porous supporting body and the upper surface of blotting membrane is adjacent to flow distributor, so that in traceDo not have bubble between flow distributor.Bubble between the two surfaces can result in no flow region.Trace retainerIt is then placed in carrier, then the carrier is securely closed with bolt lock mechanism.The equipment is placed on pressure source（Vacuum or malleation）Manifold on or in.Preferably,（It is one or more）Blotting membrane is soaked in advance.Pressure（Vacuum or justPressure）It is unlocked and the liquid of such as cleaning solution or reagent is placed in the trap of carrier.The pressure has lasted up to liquidMove through the equipment and（It is one or more）Film.Then pressure is switched off.
When using more than one blotting membrane, they can be overlapped the to each other to arrangement in series, and can be easilyThe enough liquid comprising equating expections reagent is set to move through multiple layers in a process steps.Usually, when using more thanDuring one layer, preferably people are every time using between 2 layers and 10 layers, preferably between 2 layers and 5 layers.
It can be closed in pressure source or adding liquid in the case that pressure source is only briefly opened, so that liquid enters（It is one or more）Film simultaneously is allowed to be cultivated（Such as it may require in the case of one-level or secondary antibody）.Pressure is rightIt is unlocked to remove liquid and/or replaced with another liquid then used afterwards.Preferably, during washing, vacuumOpening is left on, and then adds remaining washing lotion.
Optionally, if one want to, collection vessel 70 can be placed on below equipment by people（Fig. 9 A）, preferably in discriminationPipe in itself in or downstream, such as antibody collection tray.Then it can be used to collect one or more uncombined reagents, and it mayIt is expensive and can be collected and be recycled for using in future experiment.Vessel can also be subdivided into multiple rooms, multiple rooms withThe appropriate section of blotting membrane is aligned and is in fluid communication.
Additionally or alternatively, people can place absorbent substrate, absorbent in the downstream flow path below retainerMatrix can reversibly combine expensive one or more and be not associated with reagent.The matrix preferably takes the form of monoblock, such asPad, connector or the scraps of paper, it is positioned such that to pass through matrix by all liq of blotting membrane and retainer.Then it may be usedIt is removed and reagent is eluted or if desired, it can make the reagent of combination on the spot after the test of blotting membrane is completedIt is eluted.
Other processes can also be used together with the equipment of embodiment disclosed herein.
The film includes the one or more materials that will be detected in its space.Usually, these materials due to by fromFor electrophoresis or chromatographic solid support body trace or it is applied directly and is present in space, generally detects certain kindsThe presence of section bar material, it is not present or measures, such as antibody or specified protein, i.e., Dot-Blot types experiment as described above.However,The definition of film is not limited to these situations, and applies to film in its space comprising the one or more materials that will be detectedAny situation.It is being conceived to include being generally used to running gel for the film type used in embodiment disclosed hereinCarry out the film of trace, such as nitrocellulose, nylon or various other polymeric membranes, such as polyvinylidene fluoride（PVDF）, by horse SaZhu Sai states Billerica Millipore companies sell as IMMOBILON films.
Multiple material can be used to be replicated in the result of the running gel performed on various samples, as understood in the art's.Most commonly, sample is raw comprising independent protein, antibody, nucleic acid, oligonucleotide, complex carbohydrates class etc.Thing material, but the application of the technology is not limited to these materials.The technology can be applied to include what will be detected inside itAny film of material, the chemical composition regardless of film or target substance.
When using represent electrophoresis result duplicate film when, can by using comprising transmission buffer film, pass through electricityElution performs transmission of the material to be detected from gel to film by the dry method trace of gel.Technology for these transmissionIt is well known in the present art, and does not constitute a part for embodiment disclosed herein.
Liquid to be supplied can be included into detection reagent, or simply can be provided as washing lotion.DetectionThe property of reagent is of course depend upon substance to be detected.Generally, by between antigen and antibody or its immune response partImmune response detect protein；Generally the presence of nucleic acid fragment is detected with appropriate oligonucleotide sample.If neededWill, can be supplemented with label detection be responsible for to detect material at once or specific reaction material, and may need manySecondary application detection reagent, for example, code can include passing through supply enzyme（For example, usually, horseradish peroxidase）MarkAntibody detect antigen, and then detect that this is combined by supplying the matrix for this enzyme.When adding reagent, althoughAnd it is not preferred, but malleation donor host can be used only this component of film is exposed up to the period limited.
The method for performing embodiment disclosed herein at room temperature is most convenient, but be can also be used elevated and relatively lowTemperature.This can be by by equipment, its surrounding environment（Such as in hot tank or cooler bin）Or the liquid heating used in system comesRealize.
Then can be by the antibody previously combined being peeled off and then being used antibody or other specific target eggs of other samples from traceWhite matter subsequently cultivate and analyze trace with multiple antibody or sample with during in the equipment of the present invention.The stripping processAg-Ab key is broken, and antibody is dissolved in buffer around.This generally passes through the combination of cleaning agent and heatOr by being realized exposed to high or low pH.The equipment is combined with flow distributor makes it possible for high or low pH methods in factThe stripping of existing trace.Directly（For example, using with the same traffic distributor for stripping）Or subsequent print after storageFollow-up detect again of mark can be used and initial probe identical code.Appropriate Tool Box for bar trace can be in trade (brand) nameClaim ReBlot Plus kit（Species #2500）、ReBlot Plus-Mild solution（Species #2502）And ReBlotPlus-Strong solution（Species #2504）Under obtained from Chemicon International companies.
In standard western blotting, antigen or target are transferred to film support, and are detected with appropriate sample, such asAntibody, protein（For example, a-protein）Or hemagglutinin（Protein or glycoprotein, it is attached to carbohydrate portions）.At certainIn a little applications, reverse form is used（For example, reverse array）, wherein, antibody or other samples are placed to film or other supportsOn body（Generally in a matrix format）, and antigen or target are presented to the sessile antibody on array.Antigen or the mark of target can be passed throughNote or specifically secondary antibody realizes the visualization of target-sample binding events by using target institute.Reverse array is usually adoptedWith the mixture of target, for example, enable to realize the lysate of parallel processing with different fluorescence color markers.It also can use thisLiterary disclosed embodiment performs counter-test.